Biomedical Engineering Reference
In-Depth Information
out for at least 21 times, and SUVs are harvested at the opposite side of the
membrane to filter out large particles that might still be present. In addition to
manual extrusion, high-pressure French press can also be used to extrude the MLVs,
producing a high yield of SUVs. This method can be applied to a variety of lipids
and lipid mixtures and can be scaled-up. The extruded SUVs are always larger
than the vesicles formed via sonication [ 17 ]. It needs to be noted that all of the
extrusion/sonication operations need to be carried out above T c before SUVs are
prepared. SUVs have low encapsulation efficiency of regent since the aqueous
space per mole of phospholipids is in the range of 0.2-1.5 l (e.g., 0.1-1.0%
encapsulation). Addition of cholesterol and charged lipids in the lipid mixtures can
increase the aqueous volume. Finally, there is a limit to the size of reagent that can
be encapsulated, where the molecular weight is typically below 40,000.
To form LUVs, in addition to extrusion, reverse-phase evaporation (REV) can be
used. In this method, the lipid or lipid mixture is first dissolved in an organic solvent,
to which a buffer is added with subsequent sonication [ 21 ]. This mixture then
undergoes rotary evaporation under reduced pressure, forming a gel-like material
which spontaneously forms uniform liposomes. One of the disadvantages of the
REV method is the exposure of the encapsulating reagent to an organic solvent,
which could result in denaturation of proteins that need to be encapsulated. One of
the popular methods to form GUVs is electroformation, where an alternating electric
field is applied to a lipid film causing swelling and fluctuations in the bilayers
leading to the separation of the lamella and the formation of giant vesicles [ 22 , 23 ].
4.5
DNA Conjugation
DNA is a highly negatively charged molecule. The most straightforward method
to attach DNA is to use cationic liposomes. Taking advantage of electrostatic
attraction, polyanionic DNA and polycationic liposomes can form stable complexes,
which have been widely used for DNA transfection. In these systems, however,
the molecular recognition properties of DNA might be compromised due to the
strong electrostatic binding with liposome. To achieve more specific DNA-mediated
interactions, DNA can be covalently incorporated as a lipid head group. A number
of strategies have been developed to realize this goal as summarized below.
4.5.1
Cholesterol-Labeled DNA
One of the popular ways to attach DNA to a liposome is using a cholesterol-modified
single-stranded (ss)DNA, which spontaneously inserts itself into the hydrophobic
interior of the lipid membrane [ 24 ]. While this method is fast, since cholesterol
is a small lipid, the association of the inserted DNA is relatively weak and the
reaction is not quantitative. As a result, the number of DNA inserted into a liposome
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